Despite the "relative" ease of inducing transplant tolerance in some rodent models, the same approaches have often proven difficult to translate into larger animals and patients. One reason is that small animal models often fail to account for the barrier of memory. In addition to this known hurdle, increasing evidence indicates that inflammation (which may occur as a result of rejection, ischemia-reperfusion injury, infection etc.) can also inhibit tolerance induction. The central tenet of this project is that two events occurring very early during the process of engraftment, inflammation and antigen encounter, can determine graft outcome and tolerance susceptibility by modulating the balance between regulatory cells and effector/memory cells. Our theoretical framework is that engraftment initiates a "race" or "competition" between regulation and effector/memory development, and the result of this is crucial to the outcome of the graft. Our aims will test the hypotheses that: inflammation promotes the development of donor specific memory, while at the same time inhibiting the development of regulation (aim #1);that antigen encounter, in addition to its known effects on effector/memory development also shapes Treg development (aim #2), and that regulation which is "dysfunctional" in the context of memory, can nonetheless be harnessed to block memory responses if adjunctive approaches are use (aim #3). This work will be important for the studies in project #1, in particular those that focus on how the TIM1:TIM4 pathway effects Treg generation and conversion. .There will also be close interactions with project #3, particularly in the area of how cytokines and inflammation interact to affect memory and regulation, and how costimulatory blockade affects these processes. We bring a number of important tools that will be critical to.these studies. Through our ongoing interactions with Terry Strom and Mo Sayegh as part of the PPG, we have obtained foxp3-GFP knock-in mice. These valuable animals can be used to separately study the stability, expansion and function of naturally arising Tregs, and the conversion, expansion and function of antigen/cytokine-induced Tregs. In addition to these animals, other resources include MHC class II alloreactive TCR transgenic mice, MyD88- deficient mice, and crosses amongst all of these strains. Collectively, these studies will provide important new insights into the differentiation and fate of alloreactive T cells in vivo, define how these events are modulated by inflammation and antigen encounter, and explore approaches that will allow for the control of memory T cell responses by regulatory T cells.